The direct electrochemistry of sacrosine oxidase (SOX) immobilized on graphene, chitosan and silver nanoparticles (AgNPs) modified glassy carbon electrode was realized. SOX exhibits a pair of well-defined reversible redox peaks in 0.1 M pH 7.0 phosphate buffer solution (PBS) with the oxidation peak potential of-0.446V and the reduction peak potential of -0.496 V. The immobilized SOX retains its bioactivity, and exhibits a surface confined, reversible two-proton and two-electron transfer reaction. It also has good stability, and a fast heterogeneous electron transfer rate with the rate constant (k(s)) of 1.8 s(-1). The surface coverage of SOX is 1.18 x 10(-7) mol/cm(7). This biosensor shows excellent sensitivity towards H2O2 in 0.1 M PBS (pH 7.0) with a wide linear range from 1.0 to 177 mu M. The detection limit is 1.0 mu M. The apparent Michaelis-Menten constant for immobilized SOX is calculated to be 0.18 mM. The excellent performance of the biosensor is attributed to large specific surface area and high conductivity of graphene and AgNPs, and good biocompatibility of chitosan, which improve the enzyme absorption and promote direct electron transfer between SOX and the electrode surface. (C) 2012 Elsevier Ltd. All rights reserved.